The high-risk papillomaviruses are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that have been shown to modulate cell growth.and differentiation, target the p53 and Rb tumor suppressor proteins, and induce the hTERT gene and cellular immortalization. In addition to the E6 and E7 proteins, the high-risk papillomaviruses also encode the hydrophobic, membrane-associated E5 protein. In recent months, several studies have established that the evolution of the E5 protein is tightly linked to that of the E6 protein and that the E5 genes of high-risk papillomaviruses have characteristics that separate them from those of low-risk viruses. The high-risk HPV-16 E5 protein has a wide spectrum of biological activities, ranging from alterations of growth factor receptor turnover, MHC transport, endosome acidification, anchorage-independent growth, and intercellular junction communication. Previously we have shown that the E5 protein binds a component of the V-ATPase complex and interferes with endosome acidification, thereby giving some insight into how E5 might potentiate the signaling of growth factor receptors. In the current proposal, we have discovered additional targets for E5, including caveolin and a 116 kDa cellular protein. Based upon our accumulated knowledge regarding the detergent solubility properties of E5 and the identified E5-associated proteins, we hypothesize that E5 partitions into membrane lipid rafts and associateswith proteins that are resident in these signaling platforms. We have constructed a model that conceptually links the identified E5 targets with the plethora of known E5-induced cellular phenotypes and have designed experiments based upon this model. The specific delineation of how E5 is targeted to rafts, how it binds to specific raft-resident proteins, and how is interferes with the functions of these proteins is the focus of this grant. It is anticipated that insights into E5 functions will illuminate its linkage to HPV-induced malignancy.